Optical imaging of biological and/or chemical conditions of cells and tissues of a living subject is an area of significant importance. One of the biggest challenges in cell biology is the imaging of living cells. Conventional optical labels such as fluorescent dyes or markers are typically introduced into tissue samples to signal changing biological and/or chemical conditions of tissues. However, these conventional labels have many drawbacks. Conventional labels are generally toxic to living cells and tissues comprised of living cells. Furthermore, conventional labels such as fluorescent dyes or green fluorescent proteins (GFP) generally suffer from short-lived fluorescence because the dyes undergo photo bleaching after minutes of exposure to an excitation light source. Therefore, they are unsuitable for optical imaging that requires extended time period of monitoring. Furthermore, these labels are sensitive to environmental changes such as pH and oxygen concentration. Finally, recombinant GFP fusion proteins are cumbersome to construct, and long-term imaging (more than 3 days for example) with GFP requires the time-consuming process of establishing stable-expressing clones.
Other types of labels such as polymeric, magnetic and metallic particles have been introduced into cells. An alternative technology is the use of inorganic semiconductor nanocrystals, or quantum dots. Quantum dots, such as CdSe/ZnS core/shell nanoparticles, are inorganic fluorophores and generally have a size below 10 nm in diameter. Compared to conventional dyes, they have a much higher photobleaching threshold and negligible photobleaching under biological imaging conditions. Quantum dots can be silanized and, in that form, have reduced phototoxicity and are highly resistant to chemical and metabolic degradation.
Typically, there are three steps before quantum dots (QDs) can be employed for biological applications: 1) synthesis; 2) coating; and 3) bio-conjugation. The as synthesized QDs are generally coated with trioctylphosphine oxide (TOPO), hexadecylamine (HDA) or octadecylamine (ODA) and therefore hydrophobic in nature, and are soluble only in organic solvents.
For the design of water-soluble QDs, different coating methods are available and are accomplished either by exchange of TOPO or through hydrophobic addition reactions. TOPO ligands are often exchanged with thiol-functionalized compounds such as mercaptoacetic acid, dihydrolipoic acid, dithiothreitol, and cysteine containing peptides. The QDs have also been coated with inorganic silica or organic protective polymer layer. The coated particles are then bio-conjugated to peptides, proteins or antibodies for cellular tracking applications.
Silica and polymer coatings significantly increased the overall size to about 20 nm and restricted their use in certain imaging applications as the size of QDs determines the renal clearance of the QDs. QDs of size less 5.5 nm resulted in rapid and efficient urinary excretion and elimination from the animal body.
Additionally, during coating and bio-conjugation, the QDs tend to lose their fluorescence (up to 30-40% from the original quantum yield). The labeling of stem cells is an important area in the context of human regenerative medicine for example. However, in most of the cases, the labeling of stem cells with QDs is non-specific. Even though in vitro and in vivo imaging with QDs has been demonstrated, there remains a need to address the problems that exists in the art especially in relation to cell imaging labels associated with the use of particles such as quantum dots and nanoparticles.